Trail enhancers for the selective killing of cancer cells

ABSTRACT

The invention is directed to methods of inducing apoptosis, arresting cell cycle, or inhibiting cellular proliferation, or any combination thereof, in a tumor cell, by administration of an effective amount of an N-acyl homoserine lactone analog (AHL), optionally in conjunction with a tumor modulating agent such tumor necrosis factor (TNF) related apoptosis inducing ligand (TRAIL) to the patient. Novel bioactive analogs of an N-acyl homoserine lactone are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. provisionalapplication Ser. No. 61/803,177, filed Mar. 19, 2013, which applicationis incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under HHSN27200700038C,AI077644, AI079436, and AI094348, awarded by the National Institutes ofHealth. The U.S. government has certain rights in the invention.

BACKGROUND

Apoptosis is a genetically programmed and physiologically important formof cell death. It is a conserved cellular homeostatic mechanism withimportant roles in normal development through maintaining of cellturn-over in healthy adult tissues. Abnormal apoptotic activity has beenlinked to the pathogenesis of autoimmune and infectious disorders,including persistent inflammatory diseases (Gyrd-Hansen, et al., Nat RevCancer 10(8) 561-74). Evading apoptosis has also been identified as ahallmark of cancer (Hanahan D., et al., Cell 2000, 100(1), 57-70).Therefore, triggering apoptotic processes may be important for killingcancer cells and sensitizing them to different therapeutic regimens(Ashkenazi, A., Nat Rev Drug Discov 2008, 7(12), 1001-12; Ashkenazi, A,et al., J Clin Invest 1999, 104(2), 155-62).

Among promising candidates for cancer therapeutics is tumor necrosisfactor (TNF) related apoptosis inducing ligand (TRAIL) initiatedapoptosis through the receptor-mediated mechanism, also referred to asthe extrinsic apoptotic pathway. In contrast to the naturally occurringpro-apoptotic ligands such as TNF and Fax ligand (FasL), TRAIL infusioninto mice does not cause a lethal response or detectable toxicity totissues and organs (Ashkenazi, A., Nat Rev Drug Discov 2008, 7(12),1001-12; Ashkenazi, A, et al., J Clin Invest 1999, 104(2), 155-62;Walczak, H. et al., Nat Med 1999, 5(2), 157-63). Furthermore, thepotential significance of TRAIL for killing cancer cells has beensupported by studies in animal models demonstrating that this cytokinepossesses selective toxicity to human tumor xenografts but not normaltissues. However, sensitivity to TRAIL-induced apoptosis is a key factorlimiting the efficacy of TRAIL treatment, because a spectrum ofsensitivity is observed in different malignant cells (Lippa, M. S. etal., Apoptosis 2007, 12(8), 1465). Furthermore, similar to normal cells,some cancer cells are also resistant to TRAIL-induced apoptosis.

The increasing understanding of the molecular details of apoptosisindicates that tumor cells can acquire resistance to apoptosis throughinterference with either extrinsic or intrinsic apoptotic signalingpathways. However, most cancer cells retain the capacity to undergoapoptosis if triggered through mechanisms that can overcomeanti-apoptotic influences. For example, inhibition of NF-κB activitysignificantly increases apoptosis induced by apoptotic stimuli (Beg. A.A., et al, Science 1996, 274 (5288), 782-4; Wang, C. Y., et al., Science1996, 274 (5288), 784-7; Van Antwerp, D. J., et al., Science 1996, 274(5288), 787-9; Liu, Z. G., et al., Cell 1996, 87(3), 565-76). Inaddition, enhancing apoptosis also occurs upon activation of severalintracellular non-apoptotic signaling processes, including the JNKpathway or endoplasmic reticular (ER) stress, known in eukaryotic cellsas the unfolded protein response (UPR) (Ron, D. et al., Nat Rev Mol CellBiol 2007, 8(7), 519-29). It has been observed that UPR activators suchas tunicamycin, thapsigargin and RRR-α-tocopherol ether-link acetic acidanalog (α-TEA), senstivize cancer cells to TRAIL-inducing apoptosis(Jiang, C. C., et al., Cancer Res 2007, 67(12), 5880; Chen, L. H. etal., Carcinogenesis 2007, 28(11), 2328-36; Tiwary, R., et al., PLoS One5(7), e11865). However, these reagents induce constitutive and sustainedactivation of the UPR.

Bacterial metabolites play important roles in inflammation-mediatedprocesses essential for normal development and the pathogenesis ofnumerous chronic diseases, including cancer. Inflammation is typicallyinitiated as an innate immune response to specific bacterial productsthrough receptor-dependent mechanisms, in which induction of thetranscription factor NF-κB is required for both activation of the immunesystem and the control of apoptosis in activated cells. For example, inthe presence of Gram-negative bacteria, NF-κB activation is initiallyinduced in response to bacterial lipopolysaccharide (LPS), an agonist ofthe Toll-like receptor 4 (TLR4), leading to the expression ofNF-κB-regulated genes encoding pro-inflammatory cytokines, such as tumornecrosis factor-α (TNF) and interlekin-1 (IL-1). After the engagement ofTNF or IL-1 receptors, additional rounds of NF-κB activation amplifythis LPS-induced inflammatory response. NF-κB-dependent processes, inconcert with other signaling pathways, up-regulate the expression ofpro-apoptotic cancer immunosurveillance effectors, including theTNF-related apoptosis-inducing ligand (TRAIL), an essential mediator ofapoptotic cell death particularly in cancer cells. Although theLPS-induced inflammatory response results in the release ofpro-apoptotic cytokines such as TNF and TRAIL, cancer cells receivingthese death signals can still survive due to the suppressive effects ofNF-κB signaling on apoptosis.

SUMMARY

The invention is directed in various embodiments to method of inducingapoptosis, arrest of cell division, or inhibition of cell proliferation,or any combination thereof, in a tumor cell. Tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL) preferentially inducesapoptosis in cancer cells over normal cells; however, tumor cells maydevelop TRAIL resistance. Here we demonstrate that this resistance canbe overcome in the presence of bacterial acylhomoserine lactone (AHL)analogs or AHL-producing bacteria through the combined effect ofTRAIL-induced apoptosis and AHL-mediated inhibition of inflammationregulated by NF-κB signaling. This discovery unveils a previouslyunrecognized symbiotic link between bacteria and hostimmunosurveillance.

The invention provides, in various embodiments, a pharmaceuticalcomposition comprising an effective amount of an N-acylhomoserinelactone (AHL) analog compound of formula (I):

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15carbon atoms having one or more diazirenyl groups, optionally having oneor more carbonyl groups at positions 4 or greater on the alkyl, alkenylor alkynyl groups, and further optionally substituted with azido,hydroxyl, or halo; or a pharmaceutically acceptable salt thereof; and,optionally, a pharmaceutically acceptable excipient. For example, theAHL compound can be a compound is of formula (II)

wherein R¹ is a linear alkyl, alkenyl or alkynyl of about 7 to 13 carbonatoms, optionally having one or more of:

-   -   (a) a diazirenyl group;    -   (b) one or more carbonyl groups;    -   (c) or, one or more independently selected azido, hydroxyl, or        halo groups.

For example, the compound of formula (I) can be the (S)-enantiomer ofthe compound with respect to the chiral center at the position ofbonding of the nitrogen atom to the lactone ring.

In various embodiments, the invention provides a method of treating atumor in a patient, comprising administering to the patient an effectiveamount of an N-acylhomoserine lactone (AHL) analog compound of formula(I) as described above. Optionally, an effective amount of a tumormodulating agent such as a TRAIL polypeptide can also be administered.

In various embodiments, the invention provides a method for inducingapoptosis, arrest of cell division, or inhibition of cell proliferation,in a tumor cell, comprising contacting the cell with an effective amountof an N-acylhomoserine lactone (AHL) compound, and, optionally, aneffective amount of a tumor modulating agent, such as a TRAILpolypeptide, wherein the AHL compound is of formula (I), as describedabove.

In various embodiments, the invention provides methods of synthesis ofAHL analogs of the invention, useful for practicing methods of theinvention.

In various embodiments, the invention provides a kit comprising acompound of the invention, such as compound (S)-(3-N₂), optionallydissolved in a pharmaceutically acceptable liquid medium, in acontainer, the kit optionally further comprising a tumor modulatingagent, such as TRAIL, optionally dissolved in a pharmaceuticallyacceptable liquid medium, in a second container; further optionallycomprising dosing or storage information, or both.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: C12 Induces the UPR in mammalian cells. (A and B). Comparison ofthe macrophage responsiveness to P. aeruginosa (P.a.), S. tvphimurium(S.t.), and S. aureus (S.a.). RT-PCR assay of XBP1 (A) and Western blotanalysis (B) of p38, its phosphorylated form (p-p38) as well asphosphorylated forms of PERK (p-PERK) and eIF2α(p-eIF2α) in cellextracts after treatment with bacteria are shown. (C) Macrophages werestimulated with P. aeruginosa wild type (wt) or lasI mutant (ΔlasI), andcell extracts (total RNA or total protein) were prepared and analyzed asin (A and B). (D and E) RT-PCR assay of XBP1 or actin in total RNAprepared from macrophages after treatment with compound (C12), i.e., the(S)-enantiomer, or with the (R)-enantiomer (C12R), or the hydrolysisproduct (C12h) are shown. (F) Total RNA was prepared from lung of miceafter inoculation of vehicle (−) or (C12) and analyzed as in (D). (G)Chemical structures of compounds (C12), (C12R) and (C12h). Compound(C12) is the (S)-enantiomer of the compound of formula:

FIG. 2: C12 Induces the UPR through modulation of the sphingolipidmedabolism. (A and B) RT-PCR assays of XBP1 in total RNA prepared fromthe macrophages treated with C12, Tm or Tg in the presence or absence ofCHX (A) or D-MAPP (B) as indicated. (C) Western blot analysis shows theeffect of D-MAPP on (C12)-induced phosphorylation of eIF2α (p-eIF2α) andp38 (p-p38); Western blot for actin is a loading control. (D)Macrophages were stimulated with LPS in the presence or absence ofD-MAPP, and total protein extracts were prepared and analyzed by Westernblot for p-p38 and expression of IκBα. (E) RT-PCR assay (top panel) orWestern blot (bottom panel) monitors the levels of XBP1 or eIF2α orp-p38 in samples prepared from macrophages after treatment with (C12),sphingosine (Sph) or dimethylsphingosine (DMS). (F) TLC assay monitors³H-Sph metabolism in macrophages treated with DMSO (Vehicle) or C12 asindicated. Location of standard sphingolpids is indicated on the left.Cer, ceramide; Sph, sphingosine; S1P, sphingosine 1-phosphate; SM,sphingomyelin.

FIG. 3: Sensitivity of cancerous and non-cancerous cells to apoptosisinduced by TNF, C12 or a combination of C12 and TNF. Cells wereincubated with TNF, (C12) or a combination of TNF and (C12) asindicated, and the cleavage of PARP (an apoptotic marker) was determinedby Western blot. (A and B) Breast cancer MCF-7 cells transfected withcontrol vector (Vector) and vector expressing Bcl-2 or CLARP. (C) HeLacell line. (D) Normal human bronchial epithelial cells (NHBE).

FIG. 4: C12 sensitizes lung cancer cell line A549 to TRAIL. (A)Viability of cells was examined after 24-hour incubation in mediacontaining TRAIL (50 ng/ml), compound (C12) (1 μM), compound (C12R) (25μM) or the indicated combination of TRAIL (T) with (C12) or (C12R). Thechemical structures of (C12) and its stereoisomer (C12R) are shown onthe right. (B) Viability of cells was examined after 24-hour incubationin media containing TRAIL (50 ng/ml) and the indicated doses of C12.Viable cells remaining after the treatment were determined as a functionof mitochondrial activity of living cells according to XTT-basedtoxicology assay kit (Sigma) and are shown as a percentage of viableuntreated cells.

FIG. 5: C12 sensitizes lung cancer cell line A549 to TRAIL-inducedapoptosis. Cells were treated with TRAIL, (C12) or a combination ofTRAIL and (C12) for indicated period of time, and cellular extracts wereexamined by Western blot analysis for the apoptotic markers (the cleavedform of caspase-9, caspase-3 or PARP), the stress responses (thephosphorylated forms of eIF2α, p38 or JNK), and actin (a loadingcontrol).

FIG. 6: Primary human B cells are resistant to TRAIL in the presence ofC12. (A) Cells were treated by TRAIL (T), (C12), thapsigargin (Tg) or acombination as indicated. Viable cells remaining after a 24-hourstreatment were determined as a function of mitochondrial activity ofliving cells according to XTT-based toxicology assay kit (Sigma) and areshown as a percentage of viable untreated cells. (B) Cells were treated6 hr by TRAIL, (C12), thapsigargin (Tg) or a combination as indicated.The cellular extracts were examined by Western blot for the cleavage ofPARP as an apoptotic marker or for actin as a loading control.

FIG. 7: Sensitivity of cancerous and non-cancerous cells to acombination of TRAIL and (C12). Normal human bronchial epithelial (NHBE)and cancerous human HeLa cells were incubated with TRAIL and indicateddoses of (C12), and cell viability relative to untreated control wasdetermined by XTT assay.

FIG. 8: Safety and pro-apoptotic activity of (C12). Cells were incubatedwith TRAIL, (C12), bormetazomid (Bor) or a combination of TRAIL and(C12) or Bor as indicated, and cell viability (top panels) or thecleavage of PARP (bottom panels) was determined. (A) Lung cancer cellline A549. (B) Primary human hepatocytes from normal donor.

FIG. 9: Comparison of (C12)-mediated and compound #15-mediated effectson sensitivity of cancerous cells to TRAIL. (A) Viability of A549 cellswas examined after 24-hour incubation in media containing TRAIL (25ng/ml) and the indicated doses of (C12) or its analog (#15) (see Table1). (B) A549 cells were treated by TRAIL, tunicamycin (Tu), thapsigargin(Tg), compound #15, (C12) or a combination as indicated. The cellularextracts were examined by Western blot for the cleavage of PARP as anapoptotic marker or for actin as a loading control.

FIG. 10: Biological activity of the analogs. (A) Lung cancer A549 cellswere treated by (C12) or its analogs (compound 13, 16 and 17, seeTable 1) as indicated, and the cellular extracts were examined byWestern blot for the phosphorylated forms of eIF2α or p38 and actin as aloading control. (B) A549 cells were treated by TRAIL, compound, (C12)or a combination as indicated, and the cellular extracts were prepared(6-hour treatment) and examined by Western blot for the cleavage of PARPas an apoptotic marker as well as indicated in (A).

FIG. 11. (C12)-producing bacteria or (C12) promote cytokine-mediatedapoptosis in cancer cells. a) Chemical structures of AHLs examined inthis study. b) Lung cancer cells were incubated with or without P.aeruginosa (Bac) wild type (wt) or a lasI mutant strain (ΔlasI) in thepresence or absence of TNF or TRAIL as indicated. After 2 h, celllysates were prepared and analyzed by immunoblotting with antibodiesspecific for PARP or actin as a loading control. c) Lung cells wereuntreated (Mock) or treated with TNF or TRAIL in the presence of theindicated doses of (C12) for 6 h; cell samples were analyzed as in b).d) Comparison of lung cell responsiveness to TRAIL, (C12) or theircombination. Western blot analysis of PARP, IκBα, the phosphorylatedform of p38 (p-p38) and actin in cellular extracts prepared aftertreatment with stimuli as indicated.

FIG. 12. (C12) promotes the TRAIL-mediated killing of cancer cells. a),c) Western blot analysis of PARP cleavage in cancer or normal cellstreated for 3 h with TRAIL, (C12) or a combination of both, asindicated. b), d) XTT-based assay monitoring the viability of cancer andnormal cells grown for 18 h in media containing TRAIL and the indicateddoses of (C12). Cell survival was ˜100% in control samples (untreatedcells) as well as in samples incubated with TRAIL alone or the samedoses of (C12) without TRAIL.

FIG. 13. AHL-mediated inhibition of inflammation-induced NF-κB signalingis sufficient for rendering tumors susceptible to TRAIL-inducedapoptosis. a) Western blot analysis of PARP cleavage in cancer cellsstimulated for 3 h with TRAIL or its combination with different AHLs asindicated. b) Western blot analysis of PARP cleavage as well asphosphorylated forms of eIF2α and p38 in extracts from bonemarrow-derived macrophages (BMDM) stimulated with (C12) or (3-N₂), i.e.,(S)-(3-N₂), as indicated. c) Western blot analysis of PARP cleavage andtemporal profiles of IκBα expression in BMDM extracts prepared aftertreatment with LPS or its combination with (C12) or (3-N₂) . . . d)Inhibitory effect of (C12) and its derivatives on LPS-induced TNFproduction in BMDM. e) Lung cancer cells were exposed to (3-N₂) (10 μM),TRAIL (0.5 ng/ml), TNF (20 ng/ml) or their combinations as indicated.After 2 h, cell lysates were prepared and analyzed by Western blot forPARP cleavage, IκBα and actin.

DETAILED DESCRIPTION

Alkyl groups include straight chain and branched alkyl groups andcycloalkyl groups having from 1 to about 20 carbon atoms, and typicallyfrom 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms.Examples of straight chain alkyl groups include those with from 1 to 8carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl,n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groupsinclude, but are not limited to, isopropyl, iso-butyl, sec-butyl,t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As usedherein, the term “alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkylgroups as well as other branched chain forms of alkyl.

Alkenyl groups include straight and branched chain and cyclic alkylgroups as defined above, except that at least one double bond existsbetween two carbon atoms. Thus, alkenyl groups have from 2 to about 20carbon atoms, and typically from 2 to 12 carbons or, in someembodiments, from 2 to 8 carbon atoms. Examples include, but are notlimited to vinyl, —CH═CH(CH₃), —CH═C(CH₃)₂, —C(CH₃)═CH₂,—C(CH₃)═CH(CH₃), —C(CH₂CH₃)—CH₂, cyclohexenyl, cyclopentenyl,cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others.

Alkynyl groups include straight and branched chain alkyl groups, exceptthat at least one triple bond exists between two carbon atoms. Thus,alkynyl groups have from 2 to about 20 carbon atoms, and typically from2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms.Examples include, but are not limited to —C≡CH, —C≡C—CH₃), —C≡C—CH₂CH₃),—CH₂C≡CH, —CH₂C≡C—CH₃), and —CH₂C≡C—CH₂CH₃) among others.

When a substituent is more than monovalent, such as O, which isdivalent, it can be bonded to the atom it is substituting by more thanone bond, i.e., a divalent substituent is bonded by a double bond; forexample, a C substituted with O forms a carbonyl group, C═O, which canalso be written as “CO”, “C(O)”, or “C(═O)”, wherein the C and the O aredouble bonded. When a carbon atom is substituted with a double-bondedoxygen (═O) group, the oxygen substituent is termed an “oxo” group. Whenit is stated herein that an alkyl, alkenyl, or alkynyl group comprisesan carbonyl group, what is referred to is an alkyl, alkenyl, or alkynylchain respectively wherein a carbon atom of the chain is double bondedto an oxygen atom, e.g., of formula

wherein a wavy line indicates a point of attachment. The carbonyl groupcan also be bonded any other carbon atom of the alkyl, alkenyl, oralkynyl.

The terms “halo” or “halogen” or “halide” by themselves or as part ofanother substituent mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine.

A “haloalkyl” group includes mono-halo alkyl groups, poly-halo alkylgroups wherein all halo atoms can be the same or different, and per-haloalkyl groups, wherein all hydrogen atoms are replaced by halogen atoms,such as fluoro. Examples of haloalkyl include trifluoromethyl,1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl,perfluorobutyl, and the like.

A “hydroxyl” group is a carbon-bonded OH group.

The term “azido” refers to an N₃ group, of formula

wherein a wavy line indicates a point of attachment.

An “diazirenyl” group as the term is used herein refers to athree-membered ring including one carbon atom and two nitrogen atoms,wherein the two nitrogen atoms are double bonded to each other and eachnitrogen atom is single bonded to the carbon atom, e.g., of structure:

wherein the tetrahedral carbon atom can be bonded to two furthersubstituents. When it is stated herein that an alkyl, alkenyl, oralkynyl group comprises a diazirenyl group, what is referred to is analkyl, alkenyl, or alkynyl chain respectively wherein a saturated carbonatom of the chain is the carbon atom bonded to each of the two nitrogenatoms. For example, a C10 alkyl group comprising a diazirenyl group canbe of formula

wherein a wavy line indicates a point of attachment. The diazirenylgroup can also be bonded any other carbon atom of the alkyl, alkenyl, oralkynyl.

An “N-acylhomoserine lactone (AHL) analog” as the term is used hereincan refer to a compound of formula (I)

of the (S) absolute configuration as designated under theCahn-Ingold-Prelog system, wherein group R is a linear alkyl, alkenyl oralkynyl chain of about 9 to about 15 carbon atoms, which can incorporatediazirenyl groups, carbonyl groups, or both, and can be furthersubstituted with groups as defined above.

An example of a compound of formula (I) is compound (3-N₂)

The diazirene is bonded to the 3-carbon of the acyl chain, and thisnomenclature is used throughout.

As is apparent, a chiral center is present at the position ofbutyrolactone ring substitution. Accordingly, the compound can be ofeither configuration, but the (S)-(3-N₂) compound is preferred, whichcan variously be displayed as

without any change in the meaning of the structure; both represent the(S)-enantiomer.

There are naturally occurring AHL compounds

which can be used in various embodiments of methods of the invention forinducing apoptosis in a tumor cell, or in treating a patient withcancer.

A “salt” as is well known in the art includes an organic compound suchas a carboxylic acid, a sulfonic acid, or an amine, in ionic form, incombination with a counterion. For example, acids in their anionic formcan form salts with cations such as metal cations, for example sodium,potassium, and the like; with ammonium salts such as NH₄ ⁺ or thecations of various amines, including tetraalkyl ammonium salts such astetramethylammonium, or other cations such as trimethylsulfonium, andthe like. A “pharmaceutically acceptable” or “pharmacologicallyacceptable” salt is a salt formed from an ion that has been approved forhuman consumption and is generally non-toxic, such as a chloride salt ora sodium salt. A “zwitterion” is an internal salt such as can be formedin a molecule that has at least two ionizable groups, one forming ananion and the other a cation, which serve to balance each other. Forexample, amino acids such as glycine can exist in a zwitterionic form. A“zwitterion” is a salt within the meaning herein. The compounds of thepresent invention may take the form of salts. The term “salts” embracesaddition salts of free acids or free bases which are compounds of theinvention. Salts can be “pharmaceutically-acceptable salts.” The term“pharmaceutically-acceptable salt” refers to salts which possesstoxicity profiles within a range that affords utility in pharmaceuticalapplications. Pharmaceutically unacceptable salts may nonethelesspossess properties such as high crystallinity, which have utility in thepractice of the present invention, such as for example utility inprocess of synthesis, purification or formulation of compounds of theinvention.

Suitable pharmaceutically-acceptable acid addition salts may be preparedfrom an inorganic acid or from an organic acid. Examples of inorganicacids include hydrochloric, hydrobromic, hydroiodic, nitric, carbonic,sulfuric, and phosphoric acids. Appropriate organic acids may beselected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which include formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic,4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic,sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric,salicylic, galactaric and galacturonic acid. Examples ofpharmaceutically unacceptable acid addition salts include, for example,perchlorates and tetrafluoroborates.

Typical compositions include a compound of the invention and apharmaceutically acceptable excipient which can be a carrier or adiluent. For example, the active compound will usually be mixed with acarrier, or diluted by a carrier, or enclosed within a carrier which canbe in the form of an ampoule, capsule, sachet, paper, or othercontainer. When the active compound is mixed with a carrier, or when thecarrier serves as a diluent, it can be solid, semi-solid, or liquidmaterial that acts as a vehicle, excipient, or medium for the activecompound. The active compound can be adsorbed on a granular solidcarrier, for example contained in a sachet. Some examples of suitablecarriers are water, salt solutions, alcohols, polyethylene glycols,polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin,lactose, terra alba, sucrose, dextrin, magnesium carbonate, sugar,cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin,acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid,fatty acids, fatty acid amines, fatty acid monoglycerides anddiglycerides, pentaerythritol fatty acid esters, polyoxyethylene,hydroxymethylcellulose and polyvinylpyrrolidone. Similarly, the carrieror diluent can include any sustained release material known in the art,such as glyceryl monostearate or glyceryl distearate, alone or mixedwith a wax.

The formulations can be mixed with auxiliary agents that do notdeleteriously react with the active compounds. Such additives caninclude wetting agents, emulsifying and suspending agents, salt forinfluencing osmotic pressure, buffers and/or coloring substancespreserving agents, sweetening agents or flavoring agents. Thecompositions can also be sterilized if desired.

The route of administration can be any route which effectivelytransports the active compound of the invention to the appropriate ordesired site of action, such as oral, nasal, pulmonary, buccal,subdermal, intradermal, transdermal or parenteral, e.g., rectal, depot,subcutaneous, intravenous, intraurethral, intramuscular, intranasal,ophthalmic solution or an ointment, the oral route being preferred.

If a solid carrier is used for oral administration, the preparation canbe tableted, placed in a hard gelatin capsule in powder or pellet formor it can be in the form of a troche or lozenge. If a liquid carrier isused, the preparation can be in the form of a syrup, emulsion, softgelatin capsule or sterile injectable liquid such as an aqueous ornon-aqueous liquid suspension or solution.

Injectable dosage forms generally include aqueous suspensions or oilsuspensions which can be prepared using a suitable dispersant or wettingagent and a suspending agent Injectable forms can be in solution phaseor in the form of a suspension, which is prepared with a solvent ordiluent. Acceptable solvents or vehicles include sterilized water,Ringer's solution, or an isotonic aqueous saline solution.Alternatively, sterile oils can be employed as solvents or suspendingagents. Preferably, the oil or fatty acid is non-volatile, includingnatural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

For injection, the formulation can also be a powder suitable forreconstitution with an appropriate solution as described above. Examplesof these include, but are not limited to, freeze dried, rotary dried orspray dried powders, amorphous powders, granules, precipitates, orparticulates. For injection, the formulations can optionally containstabilizers, pH modifiers, surfactants, bioavailability modifiers andcombinations of these. The compounds can be formulated for parenteraladministration by injection such as by bolus injection or continuousinfusion. A unit dosage form for injection can be in ampoules or inmulti-dose containers.

The formulations of the invention can be designed to provide quick,sustained, or delayed release of the active ingredient afteradministration to the patient by employing procedures well known in theart. Thus, the formulations can also be formulated for controlledrelease or for slow release.

Compositions contemplated by the present invention can include, forexample, micelles or liposomes, or some other encapsulated form, or canbe administered in an extended release form to provide a prolongedstorage and/or delivery effect. Therefore, the formulations can becompressed into pellets or cylinders and implanted intramuscularly orsubcutaneously as depot injections. Such implants can employ known inertmaterials such as silicones and biodegradable polymers, e.g.,polylactide-polyglycolide. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides).

For nasal administration, the preparation can contain a compound of theinvention, dissolved or suspended in a liquid carrier, preferably anaqueous carrier, for aerosol application. The carrier can containadditives such as solubilizing agents, e.g., propylene glycol,surfactants, absorption enhancers such as lecithin (phosphatidylcholine)or cyclodextrin, or preservatives such as parabens.

For parenteral application, particularly suitable are injectablesolutions or suspensions, preferably aqueous solutions with the activecompound dissolved in polyhydroxylated castor oil.

Tablets, dragees, or capsules having talc and/or a carbohydrate carrieror binder or the like are particularly suitable for oral application.Preferable carriers for tablets, dragees, or capsules include lactose,corn starch, and/or potato starch. A syrup or elixir can be used incases where a sweetened vehicle can be employed.

A typical tablet that can be prepared by conventional tablettingtechniques can contain:

Core: Active compound (as free compound or salt thereof) 250 mg Colloidal silicon dioxide (Aerosil) ® 1.5 mg Cellulose, microcryst.(Avicel) ®  70 mg Modified cellulose gum (Ac-Di-Sol) ® 7.5 mg Magnesiumstearate Ad. Coating: HPMC approx.   9 mg *Mywacett 9-40 T approx. 0.9mg *Acylated monoglyceride used as plasticizer for film coating.

A typical capsule for oral administration contains compounds of theinvention (250 mg), lactose (75 mg) and magnesium stearate (15 mg). Themixture is passed through a 60 mesh sieve and packed into a No. 1gelatin capsule. A typical injectable preparation is produced byaseptically placing 250 mg of compounds of the invention into a vial,aseptically freeze-drying and sealing. For use, the contents of the vialare mixed with 2 mL of sterile physiological saline, to produce aninjectable preparation.

The compounds of the invention can be administered to a mammal,especially a human in need of such treatment, prevention, elimination,alleviation or amelioration of a malcondition. Such mammals include alsoanimals, both domestic animals, e.g. household pets, farm animals, andnon-domestic animals such as wildlife.

The compounds of the invention are effective over a wide dosage range.For example, in the treatment of adult humans, dosages from about 0.05to about 5000 mg, preferably from about 1 to about 2000 mg, and morepreferably between about 2 and about 2000 mg per day can be used. Atypical dosage is about 10 mg to about 1000 mg per day. In choosing aregimen for patients it can frequently be necessary to begin with ahigher dosage and when the condition is under control to reduce thedosage. The exact dosage will depend upon the activity of the compound,mode of administration, on the therapy desired, form in whichadministered, the subject to be treated and the body weight of thesubject to be treated, and the preference and experience of thephysician or veterinarian in charge.

Generally, the compounds of the invention are dispensed in unit dosageform including from about 0.05 mg to about 1000 mg of active ingredienttogether with a pharmaceutically acceptable carrier per unit dosage.

Usually, dosage forms suitable for oral, nasal, pulmonal or transdermaladministration include from about 125 μg to about 1250 mg, preferablyfrom about 250 μg to about 500 mg, and more preferably from about 2.5 mgto about 250 mg, of the compounds admixed with a pharmaceuticallyacceptable carrier or diluent.

Dosage forms can be administered daily, or more than once a day, such astwice or thrice daily. Alternatively dosage forms can be administeredless frequently than daily, such as every other day, or weekly, if foundto be advisable by a prescribing physician.

DETAILED DESCRIPTION

We examined the Gram-negative bacteria Pseudomonas aeruginosa, anopportunistic pathogen that is only able to promote infection in hostswith defective immune system functions (Smith, R. S. and Iglewski, B. H.(2003) Pseudomonas aeruginosa quorum sensing as a potentialantimicrobial target. J Clin Invest 112, 1460-5). As compound (C12)

is a signaling molecule produced in abundance by this bacteria, weexamined whether wild type P. aeruginosa or a mutant strain lackinglasI, the gene responsible for the synthesis of compound (C12), couldrender lung cancer cells susceptible to TNF- or TRAIL-induced cleavageof poly(ADP-ribose) polymerase (PARP), an indicative characteristic ofapoptosis. Excitingly, PARP cleavage was only detected when cellsreceived a combination of TRAIL and wild type bacteria (FIG. 11b ),suggesting that compound (C12) was required for TRAIL-induced apoptosisin cancer cells. Notably, similar results were observed when otherAHL-producing bacteria were added to cytokine-stimulated cells, whilebacteria that do not possess AHL synthases had no effect.

Furthermore, titration experiments confirmed that the direct addition ofC12 or several naturally occurring AHL analogues resulted in a strongpro-apoptotic response to TNF or TRAIL, and in agreement with ourbacterial experiments, the cells were more sensitive to TRAIL (FIG. 11c).

Since activation of NF-κB signaling inhibits apoptosis, the observeddifference in the pro-apoptotic effects of TNF and TRAIL might be linkedto the distinct ability of these cytokines to modulate NF-κB activity.Consistent with this interpretation, Western blot analysis for thedegradation and re-synthesis of IκBα, an indicative biochemical markerof NF-κB signaling, revealed a robust activation of NF-κB signaling inresponse to TNF but not to TRAIL. Although no modulation of NF-κB orapoptotic signaling was induced in response to TRAIL or compound (C12),substantial changes in the levels of IκBα were matched with PARPcleavage in lung cancer cells stimulated with a combination of compound(C12) and TRAIL (FIG. 11d ). Interestingly, we also observed that thecombined action of TRAIL and compound (C12) resulted in a prolongedactivation of the mitogen-activated protein kinase (MAPK) p38 asdetermined by Western blot analysis for the phosphorylated form of p38(FIG. 11d ; p-p38). These findings suggest that compound (C12) enhancesTRAIL's ability to execute apoptosis in cancer cells through modulationof NF-κB, p38 or both signaling processes.

Despite the expression of TRAIL receptors, normal cells are resistant toTRAIL-induced apoptosis. Similar to non-transformed cells, manymalignant cells are not sensitive or only partially sensitive to thepro-apoptotic action of TRAIL. Therefore, in order to assess theselectivity of compound (C12) as a modulator of TRAIL-dependent tumorimmunosurveillance, we compared the sensitivity of several cancer celllines and normal cells to TRAIL and compound (C12). Consistent with ourprevious observations, substantial induction of PARP cleavage wasobserved in lung, colon and breast cancer cells stimulated with acombination of compound (C12) and TRAIL. In contrast, human hepatocytesfrom normal donors as well as other primary cells from normal tissueswere resistant to the same treatment (FIGS. 12a and 12c ). Importantly,longer treatment of cancer cells with TRAIL plus compound (C12)significantly decreased their viability although no effect on thesurvival of normal cells was noted (FIGS. 12b and 12d ).

While these data demonstrate a therapeutic potential of compound (C12)as an enhancer of TRAIL-dependent anticancer activity, questionsconcerning (C12)-mediated pro-apoptotic effects on immune cells, such asprimary macrophages, were still required to be addressed. Consideringthe potential inherent pharmacokinetic liabilities associated with C12,a small series of compound (C12) analogues was tested against bonemarrow-derived macrophages and TRAIL-treated lung cancer cells. Fromthese studies, an AHL lacking the 3-oxo moiety was found to becompletely inactive in both assays; however, a 3-diazirine-containingderivative of compound (C12) termed compound (3-N₂)

the (S)-enantiomer of which is referred to, wherein a diazirine ring(i.e., containing a double bond between the two nitrogen atoms) replacesthe carbonyl group of compound (C12); (see the Examples, below, forstructure, synthesis and characterization data) was found to be nontoxicto macrophages, yet exhibited toxicity against TRAIL-treated cancercells in a manner similar to the parental molecule. Moreover, we alsoobserved a comparable effect of compound (C12) and compound (3-N₂) onTRAIL-induced PARP cleavage in TRAIL resistant cancer cells (FIG. 13a ).

To better define the structure-activity relationship betweenAHL-mediated effects on the pro-apoptotic action of TRAIL and theagonistic potential of (C12) or compound (3-N₂), the responses ofmacrophages to these compounds were examined by Western blot analysisfor PARP cleavage, activation of p38, and the phosphorylation of theeukaryotic translation initiation factor 2α (eIF2α), a distinct featureof mammalian cell activation in response to C12 and its 3-oxo-analogues.A comparison of the agonistic activities of compound (C12) and compound(3-N₂) revealed that both compounds induced eIF2α phosphorylation in asimilar fashion; however, compound (3-N₂) did not induce p38phosphorylation and PARP cleavage (FIG. 13b ), suggesting thatactivation of the p38 pathway promotes AHL-induced apoptosis in additionto stimulating phagocytic activity, as previously disclosed.

Besides its agonistic activities, compound (C12) also inhibitsinflammatory responses to TNF, LPS and other TLR ligands in a widevariety of cell types. In macrophages, the anti-inflammatory activity ofC12 interferes with the inducible expression of NF-κB target genes, suchas IκBα and TNF. Our results also suggest that AHL-mediated disruptionof IκBα-dependent NF-κB signaling renders cancer cells susceptible toTNF- and TRAIL-induced apoptosis (see FIG. 11d ). Therefore, to examinewhether compound (3-N₂) affects stimulus-induced NF-κB signaling, wecompared the dynamics of IκBα expression in macrophages activated by LPSor its combination with compound (C12) or compound (3-N₂). Curiously,although the expected pro-apoptotic cleavage of PARP was observed in thepresence of compound (C12) but not compound (3-N₂), both compounds wereequally effective in blocking LPS-induced re-synthesis of IκBα (FIG. 13c). Moreover, similarity between the anti-inflammatory activities ofcompound (C12) and compound (3-N₂) were also evident from comparison oftheir inhibitory effects on LPS-induced production of TNF (FIG. 13d ).These experiments indicate that compound (3-N₂) is nontoxic to restingor inflammation-activated macrophages; however, it retains the abilityof C12 to modulate LPS-induced NF-κB signaling.

TNF is a key inflammatory mediator responsible for LPS-induced tumorgrowth, and the growth-promoting activity of TNF is dependent on NF-κBactivation. Most importantly, experiments using a mouse model ofLPS-induced tumor growth suggest that inhibition of TNF-mediated NF-κBsignaling in cancer cells converts inflammation-induced tumor growth toinflammation-induced tumor regression mediated by endogenous TRAIL. Toaddress whether an AHL is able to enhance the anti-cancer activity ofTRAIL in inflammation-activated cancer cells, we examined the effect ofa suboptimal concentration of TRAIL on PARP cleavage in cancer cellstreated with a combination of TNF and compound (3-N₂). Western blotanalysis revealed that TRAIL-dependent PARP cleavage was manifested incancer cells stimulated with TNF in the presence of compound (3-N₂), andsignificantly, the TRAIL-mediated apoptotic response coincided withdisruption of TNF-induced NF-κB signaling (FIG. 13e ).

In sum, the identification of compound (C12) and its analogues such ascompound (3-N₂) as “TRAIL-enhancers” and the ability of these compoundsto inhibit pro-inflammatory responses through modulation of NF-κBsignaling provide a proof-of-principle application for the selectivekilling of cancer cells. Notably, the synergistic effects of compound(3-N₂) on TRAIL-induced apoptosis in cancer cells were comparable withthose for an anti-cancer agent bortezomib; however, in contrast tobortezomib, compound (3-N₂) alone or in combination with TRAIL wasnon-toxic to human hepatocytes derived from tissues of normal donors. Alinkage of cancer and inflammation suggests a substantial benefit can begained from using anti-inflammatory agents, such as inhibitors of NF-κB,in cancer therapy and prevention.

Accordingly, in various embodiments, the invention provides apharmaceutical composition comprising an effective amount of anN-acylhomoserine lactone (AHL) analog compound of formula (I):

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15carbon atoms having one or more diazirenyl groups bonded thereto,optionally having one or more carbonyl groups at positions 4 or greateron the alkyl, alkenyl or alkynyl groups, and further optionallysubstituted with azido, hydroxyl, or halo; or a pharmaceuticallyacceptable salt thereof; and, optionally, a pharmaceutically acceptableexcipient.

In reference to the statement about optionally having one or morecarbonyl groups at positions 4 or greater on the alkyl, alkenyl oralkynyl groups, the position number (e.g., 4) refers to the carbonnumber of the acyl chain to which the oxygen atom is bonded (e.g., atposition 4 of the acyl chain). The acyl chain forms an amide bond withthe homoserine lactone amino group to provide the AHL compound.

For example, the AHL compound can be of formula (II)

wherein R¹ is a linear alkyl, alkenyl or alkynyl of about 7 to 13 carbonatoms, optionally having one or more of:

(d) a diazirenyl group;

(e) one or more carbonyl groups; or

(f) one or more independently selected azido, hydroxyl, or halo groups.

In various embodiments, a pharmaceutical composition of the inventioncan further comprising a tumor modulating agent that is a TRAILpolypeptide.

More specifically, the N-acylhomoserine lactone (AHL) analog is selectedfrom the group consisting of:

In various embodiments, the invention provides a method of treating atumor in a patient, comprising administering to the patient an effectiveamount of an N-acylhomoserine lactone (AHL) analog compound of formula(I)

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15carbon atoms having one or more diazirenyl groups, optionally having oneor more carbonyl groups, and further optionally substituted with azido,hydroxyl, or halo; or a pharmaceutically acceptable salt; andoptionally, a pharmaceutically acceptable excipient. For instance, thecompound can be a compound of formula (II)

wherein R¹ is a linear alkyl, alkenyl or alkynyl of about 7 to 13 carbonatoms, optionally having one or more of:

(a) a diazirenyl group;

(b) one or more carbonyl groups; or

(c) one or more independently selected azido, hydroxyl, or halo groups.

The method of the invention can further comprise administering aneffective amount of a tumor modulating agent that is a TRAILpolypeptide.

More specifically the N-acylhomoserine lactone (AHL) analog can beselected from the group consisting of:

For example, the tumor that can be treated according to a method of theinvention can be selected from the group consisting of lung, cervical,breast, and brain tumors.

The invention provides, in various embodiments, a method for inducingapoptosis, arrest of cell division, or inhibition of cell proliferation,in a tumor cell, comprising contacting the cell with an effective amountof an N-acylhomoserine lactone (AHL) compound, and an effective amountof a TRAIL, wherein the AHL compound is of formula (I):

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15carbon atoms having one or more diazirenyl groups, optionally having oneor more carbonyl groups, and further optionally substituted with azido,hydroxyl, or halo. For example, in various embodiments, the AHL analogin the effective amount is an activator of the unfolded protein response(UPR), of endoplasmic reticulum swelling (ERS), or both, in the tumorcell.

More specifically, for practice of a method of the invention, the AHLcompound is a compound of formula (II)

wherein R¹ is a linear alkyl, alkenyl or alkynyl of about 7 to 13 carbonatoms, optionally having one or more of:

(a) a diazirenyl group;

(b) one or more carbonyl groups; and

(c) one or more independently selected azido, hydroxyl, or halo groups.

More specifically, the N-acylhomoserine lactone (AHL) analog can beselected from the group consisting of:

The tumor cell can be from a tumor selected from the group consisting oflung, cervical, breast, and brain tumors.

The present invention further provides, in various embodiments, acompound of formula (I):

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15carbon atoms having one or more diazirenyl groups, optionally having oneor more carbonyl groups at positions 4 or greater on the alkyl, alkenylor alkynyl groups, and further optionally substituted with azido,hydroxyl, or halo; or, a pharmaceutically acceptable salt thereof. Forexample, the compound can be a compound of formula (II)

wherein R¹ is a linear alkyl, alkenyl or alkynyl of about 7 to 13 carbonatoms, optionally having one or more of:

(a) a diazirenyl group;

(b) one or more carbonyl groups; or

(c) one or more independently selected azido, hydroxyl, or halo groups.

The compound of formula (I) as described above can be provided in apharmaceutical combination with a tumor modulating agent that is a TRAILpolypeptide. For example, the N-acylhomoserine lactone (AHL) analog ofthe invention can be selected from the group consisting of:

In various embodiments, the invention provides a method of inducingapoptosis in a cell comprising contacting the cell with an effectiveamount of the N-acylhomoserine lactone (AHL) analog as described above,provided the AHL analog is not of formulas

Optionally, the method of inducing apoptosis further comprisesadministering an effective amount of a tumor modulating agent, such asTRAIL. The method of inducing apoptosis can be selective for a cancercell with respect to a normal cell.

In various embodiments, the invention provides a method of treating atumor in a patient, comprising administering to the patient an effectiveamount of an N-acylhomoserine lactone (AHL) analog, plus an effectiveamount of a tumor modulating agent, such as TRAIL. In variousembodiments, the AHL analog is not of formulas

In various embodiments, the method of treating a tumor can provideselective killing of cells of the tumor in the presence of normal cellsof the patient's body.

The compound of formula (I) can be a compound of formula (3-N₂)

For example, the compound of formula (I) can be the (S)-enantioner of acompound of formula (3-N₂)

In various embodiments, the invention provides a compound of formula (I)

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15carbon atoms, comprising one or more diazirenyl group, optionallyfurther comprising one or more a carbonyl group, and further optionallysubstituted with azido, hydroxyl, or halo, or a pharmaceuticallyacceptable salt thereof, provided that the compound is not

In various embodiments, the invention provides a compound of formula(3-N₂)

wherein the diazirine ring, containing a double bond between the twonitrogen atoms, replaces the carbonyl group of the compound C12 toprovide compound (3-N₂).

In various embodiments, the invention provides a pharmaceuticalcomposition comprising a compound of the invention, and apharmaceutically acceptable excipient.

In various embodiments, the invention provides a kit comprising acompound of the invention, optionally dissolved in a pharmaceuticallyacceptable liquid medium, in a container; the kit optionally furthercomprising a tumor modulating agent, such as TRAIL, optionally dissolvedin a pharmaceutically acceptable liquid medium, in a second container;further optionally comprising dosing or storage information, or both.

More specifically, the N-acylhomoserine lactone (AHL) analog of formula(I) as disclosed and claimed herein can be any of:

or any pharmaceutically acceptable salt thereof.

The tumor can be a lung tumor, a cervical tumor, breast cancer, braincancer, or the like. In various embodiments, the AHL analog of formula(II) can be in an effective amount an activator of the unfolded proteinresponse (UPR), of endoplasmic reticulum swelling (ERS), or both, in thetumor cell.

In various embodiments, the invention provides a method of inducing anunfolded protein response (UPR) in a mammalian cell comprisingcontacting the cell with an effective amount of an AHL or analog thereofof formula (I)

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15carbon atoms, comprising one or more diazirenyl group, optionallyfurther comprising one or more a carbonyl group, and further optionallysubstituted with azido, hydroxyl, or halo, or a pharmaceuticallyacceptable salt thereof.

In various embodiments, the UPR thus induced can produce cell cyclearrest and/or can inhibit cell division or proliferation. Cell cyclearrest and/or inhibition of cell division or proliferation can be aneffective therapy in treating neoplasms such as tumors. Accordingly, invarious embodiments, the invention provides a method of treating amalcondition in a patient wherein induction of the UPR, arrest of celldivision and/or inhibition of cell proliferation is medically indicated,comprising administering to the patient an effective amount of an AHL oranalog thereof of formula (I)

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15carbon atoms, comprising one or more diazirenyl group, optionallyfurther comprising one or more a carbonyl group, and further optionallysubstituted with azido, hydroxyl, or halo, or a pharmaceuticallyacceptable salt thereof.

The method of treatment comprising induction of the UPR can furthercomprise co-administration of an effective amount or concentration of aTRAIL. For example, the malcondition can comprise cancer or aprecancerous condition or tissue hyperplasia.

The invention further provides, in various embodiments, a pharmaceuticalcomposition comprising an effective amount of an N-acylhomoserinelactone (AHL) analog from a compound of formula (I):

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15carbon atoms having one or more diazirenyl groups, optionally having oneor more carbonyl groups at positions 4 or greater on the alkyl, alkenylor alkynl groups, and further optionally substituted with azido,hydroxyl, or halo; and a pharmaceutically acceptable salt. For example,the compound can be a compound of formula (II)

wherein R¹ is a linear alkyl, alkenyl or alkynyl of about 7 to 13 carbonatoms, optionally having:

(g) a diazirenyl group;

(h) one or more carbonyl groups; and

(i) optionally substituted with azido, hydroxyl, or halo.

In various embodiments, the invention can further provide thepharmaceutical composition as described above, further comprising atumor modulating agent that is a TRAIL polypeptide.

More specifically, the invention provides the pharmaceutical compositionas described above, wherein the N-acylhomoserine lactone (AHL) analog isselected from the group consisting of:

In various embodiments, the invention provides a method of treating atumor in a patient, comprising administering to the patient an effectiveamount of an N-acylhomoserine lactone (AHL) analog comprising a compoundof formula (I)

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15carbon atoms having one or more diazirenyl groups, optionally having oneor more carbonyl groups, and further optionally substituted with azido,hydroxyl, or halo; anda pharmaceutically acceptable salt. For example, to practice a method ofthe invention, the compound can be a compound of formula (II)

wherein R¹ is a linear alkyl, alkenyl or alkynyl of about 7 to 13 carbonatoms, optionally having:

(d) a diazirenyl group;

(e) one or more carbonyl groups; and

(f) optionally substituted with azido, hydroxyl, or halo.

For example, the method of treating a tumor in a patient as describedabove can further comprise administering a tumor modulating agent thatis a TRAIL polypeptide. for instance, for practicing the method oftreating a tumor in a patient as described above, the N-acylhomoserinelactone (AHL) analog can be selected from the group consisting of:

For example the tumor can be selected from the group consisting of lung,cervical, breast, and a brain tumors.

The invention further provides, in various embodiments, a compound offormula (I):

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15carbon atoms having one or more diazirenyl groups, optionally having oneor more carbonyl groups at positions 4 or greater on the alkyl, alkenylor alkynl groups, and further optionally substituted with azido,hydroxyl, or halo; and a pharmaceutically acceptable salt. For example,the compound can be a compound of formula (II)

wherein R¹ is a linear alkyl, alkenyl or alkynyl of about 7 to 13 carbonatoms, optionally having:

(d) a diazirenyl group;

(e) one or more carbonyl groups; and

(f) optionally substituted with azido, hydroxyl, or halo.

The compound can be combined with a tumor modulating agent that is aTRAIL polypeptide. The compound of the invention can be selected fromthe group consisting of:

The invention further provides, in various embodiments, a method forinducing apoptosis, arrest of cell division, or inhibition of cellproliferation, in a tumor cell, comprising contacting the cell with aneffective amount of an N-acylhomoserine lactone (AHL) compound, and aneffective amount of a TRAIL, wherein the compound is from formula (I):

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15carbon atoms having one or more diazirenyl groups, optionally having oneor more carbonyl groups, and further optionally substituted with azido,hydroxyl, or halo. More specifically, the for practice of the method forinducing apoptosis, arrest of cell division, or inhibition of cellproliferation, in a tumor cell, the AHL analog in the effective amountcan be an activator of the unfolded protein response (UPR), ofendoplasmic reticulum swelling (ERS), or both, in the tumor cell.

For instance, for practice of a method of the invention as describedabove, the compound can be a compound of formula (II)

wherein R¹ is a linear alkyl, alkenyl or alkynyl of about 7 to 13 carbonatoms, optionally having:

(d) a diazirenyl group;

(e) one or more carbonyl groups; and

optionally substituted with azido, hydroxyl, or halo.

More specifically, for practice of the method for inducing apoptosis,arrest of cell division, or inhibition of cell proliferation, in a tumorcell, the N-acylhomoserine lactone (AHL) analog can be selected from thegroup consisting of:

In various embodiments, the invention provides a kit comprising a AHLanalog, or an compound of the invention, optionally dissolved in apharmaceutically acceptable liquid medium, in a container; the kitoptionally further comprising a tumor modulating agent, optionallydissolved in a pharmaceutically acceptable liquid medium, in a secondcontainer; further optionally comprising dosing or storage information,or both.

Biological Studies

Previous work by the inventors here have shown that bacterialN-(3-oxo-acyl) homoserine lactones (AHLs) transiently induce distentionof the ER and appearance of a biochemical marker of the UPR, such asphosphorylation of eukaryotic translation initiation factor 2α (eIF2α),suggesting roles of AHLs in the UPR (V. Kravchenko, et al., J Biol Chem2006, 281(39), 28822-30). Since the inositol-requiring protein 1(IRE1)-mediated splicing of the mRNA encoding the transcription factorXBP1 (X-box binding protein) is an evolutionary conserved signature ofthe UPR (Schroder, M., et al, Ann Rev Biochem 2005, 74, 739-89), weaddressed the role of AHLs in activation of the UPR by investigating ifthe spliced form of XBP1 mRNA (sXBP1) is generated in response to thepresence of N-(3-oxo-acyl-dodecanoyl) homoserine lactone (“C12”). AnRT-PCR assay revealed that compound C12 as well as other AHLs inducedthe generation of sXBP1 in mouse embroyonic fibroblasts (MEFs) and theresponse to C12 was comparable with the effects of tunicamycin (Tg) andthapsagargin (Tg). This response to C12 was not limited to MEFs, but wasalso observed in a wide variety of cell types, including humantransformed cells.

The bacterial super community, also referred to as the microbiota,coexists with multi-cellular organisms and is the ancient environmentfactor with important roles in numerous stress-sensing physiologicalprocesses, particularly those involved in metabolic and immune responsesof the host cells. For example, the gut microbiota beneficiallycontributes to both the metabolic system by promoting the harvest ofdietary nutrients^(1, 2) and the immune system by maintaining the normallow-grade level of inflammation²⁻⁴. Traditionally, inflammatory processis required for tissue repair and involves integration of many complexsignaling mechanisms including modulation of the Toll-like receptor(TLR) pathways in response to bacterial products such aslipopolysaccharide (LPS) and peptidoglycan^(4, 5). On the other hand,the microbiota-mediated inflammation is also associated with metabolicdisorders such as obesity and cancer development^(2, 6-8). Although themolecular mechanisms underlining these disorders are inextricableinterconnected with activation of the endoplasmic reticulum (ER) stresspathway^(5, 9, 10), the bacterial component(s) triggering ER stress hasyet to be identified.

Among key biochemical signatures of the multicomponent ER stresspathway, known as the unfolded protein response (UPR), are thephosphorylation of the α-subunit of eukaryotic translation initiationfactor-2 (eIF2α) and activation of the transcription factor XBP1/Hac1(X-box binding protein-1 in metazoans and homologous to ATF/CREB inyeast, respectively)¹¹. The ability of certain Gram-negativebacterium-derived molecules, such as N-(3-oxo-acyl) homoserine lactones(3-oxo-AHLs), to induce eIF2α phosphorylation¹² as well as the presenceof 3-oxo-AHLs in samples of the microbiota¹³⁻¹⁵ prompted us toinvestigate their role in activation of the UPR. The Gram-negativePseudomonas aeruginosa and Gram-positive Staphylooccus aureus bacteriawere chosen as two evolutionary distant microbes that can form part ofhuman microbiota, and activate mammalian cells mainly through TLR4 andTLR2, respectively^(4, 16); unlike to S. aureus, P. aeruginosa alsorepresents a class of bacteria synthesizing a prototypic member of the3-oxo-AHL family, N-(3-oxo-dodecanoyl) homoserine lactone compound(C12). Additionally, the Gram-negative bacterium Salmonella typhimuriumwas used as a control microbe that contains TLR4 ligands similar tothose in P. aeruginosa, but does not synthesize compound (C12)¹⁷. Toinitiate our studies, we exposed bone marrow-derived macrophages (BMDM)to these bacteria, and macrophage responsiveness was analyzed forbiochemical markers of the UPR and the phosphorylation of themitogen-activated protein kinase p38, as a common marker of TLRpathways. Rapid and robust generation of the spliced form of XBP1 mRNA(sXBP1) was observed in response to P. aeruginosa, whereas two otherbacteria induced very faint, if any, level of sXBP1 (FIG. 1A). Moreover,although all three bacteria induced similar levels of p38phosphorylation (p-p38), additional signs of the UPR, such as thephosphorylated forms of eIF2α and PERK [the double-strandedRNA-dependent protein kinase (PRK)-like ER kinase], were only observedin response to P. aeruginosa (FIG. 1B). Notably, we found that P.aeruginosa deficient in lasI, the gene responsible for compound (C12)synthesis, lost the ability to activate sXBP1 and eIF2α phosphorylation(FIG. 1C), providing evidence that the observed induction of the UPRmarkers was linked to the presence of C12 in wild type P. aeruginosa.Indeed, direct addition of C12 resulted in time- and dose-dependentinduction of sXBP1 (FIGS. 1D and 1E). Such activation of the UPR wasselective to the natural (S)-form of compound (C12), whereas anunnatural (R)-stereoisomer (C12R) or a hydrolyzed form of compound(C12), (C12h), was ineffective (FIGS. 1D and 1G).

Importantly, the similar requirements in the structural integrity of thelactone ring motif were also evident in response to other naturalstereoisomer of the AHL family (Table 1). These effects of AHLs on theUPR activation were not limited to macrophages, but were also observedin other cell types including mouse embryonic fibroblasts (MEF), normalhuman bronchial epithelial cells and human TPH1 cell line (Table 2).More importantly, administration of C12 into mouse lung resulted in thegeneration of sXBP1 (FIG. 1F), consistent with our experiments incultured cells. Thus, AHLs represent the first example of secretedbacterial molecules that possess the ability to induce the UPR invarious mammalian cells.

Modulation of the Sphingolilid Metabolism by C12 Results in Activationof the UPR.

The unfolded protein response relies on both protein and lipidcomponents of the ER¹¹. To clarify whether C12 targets protein componentof the ER, we examined the effect of a general protein synthesisinhibitor CHX on sXBP1 induction in response to C12, tunicamycin (Tm, aninhibitor of protein glycosylation affecting the folding of newlysynthesized protein) or thapsigargin (Tg, an inducer of passive releaseof calcium from ER stores resulting to activation of the UPRindependently from de novo protein synthesis). Compared to Tg andespecially to Tm, C12-induced generation of sXBP1 was completelyresistant to CHX (FIG. 2A), suggesting that a protein component of theER stress is dispensable for the C12-induced UPR activation.

It has been suggested that the biosynthesis of lipids and regulation ofthe UPR are linked, although the specific lipid components involved inmodulation of the UPR have not defined yet^(10, 11, 18). Since de novosynthesis of the core sphingolipid, N-acyl-sphingosine or ceramide(Cer), occurs in the ER¹⁰, we sought to test whether pharmacologicalinhibitors of sphingolipid metabolism alter the UPR activation by C12.The results of these experiments identified D-MAPP, an inhibitor of Cercleavage, as an agent that substantially impaired C12-inducedphosphorylation of eIF2α(FIG. 2B). The inhibitory effect was specific toC12-mediated UPR activation, as both Tm- and Tg-mediated induction ofsXBP1 was unchanged in the presence of D-MAPP (FIG. 2C); additionally,LPS-induced activation of p38 phosphoylation and the profiles of IκBαexpression were also unaltered by D-MAPP (FIG. 2D). Thus, these findingssuggest that both IRE1 and PERK arms of the UPR sense and respond tochanges in sphingolipid metabolism induced by C12.

The backbone component of all sphingolipids, sphingosine (Sph), is aproduct of Cer cleavage. Acylation of the Sph amino group with fattyacids recycles Cer, whereas the phosphorylation of Sph results inproduction of spingosine 1-phosphate (S1P), hydrolysis of whichrepresents the final step of the metabolic pathway^(10, 19). Sph alsocan be N-methylated to N,N-dimethylsphingosine (DMS) that acts as anaturally occurred inhibitor of the sphingosine kinase activity; and itsaddition to the cells results in a decrease in S1P and accompanies withsmall increase in Sph levels^(20, 21). Toward this end, our data showedthat modulation of the Sph metabolism was correlated with the UPRactivation, because the expected increase in intracellular Sph level dueto DMS treatment resulted in the induction of sXBP1 andeIF2αphosphorylation, whereas the extracellular addition of Sph had noeffect on these biochemical markers of the UPR (FIG. 2E). Therefore, toaddress whether C12 modulates metabolic conversion of intracellular Sph.BMDM were radiolabeled with ³H-Sph in the absence or presence of C12,and the distribution of radioactivity within the sphingolipid fractionswas estimated by thin layer chromatography (TLC). We found thatsubstantial amount of ³H-radioactivity was rapidly (within 5 min)incorporated into the Cer fraction, and two hours later, it wasdistributed between the ceramide and sphingomyelin (SM) fractions.Addition of C12 dramatically changed the early, as well as the latesteps of sphingolipid metabolism; after 2 hr the expected incorporationinto the sphingomyelin was not observed but rather the radioactivityremained in the sphingosine fraction (FIG. 2F). Moreover, a 5-mintreatment with C12 also resulted in the incorporation of³H-radioactivity into another fraction (presumably S1P) that diminishedover time. Although further detailed studies of mechanisms involved inthese processes are needed, these finding provide a first piece ofevidence that C12 possesses the ability to modulate the metabolism ofsphingolipids, perhaps through the steps involved in Sph turnover andgeneration, and this effect of C12 correlates with activation of theUPR. Thus, we postulate that C12 induces cell activation throughmodulation of the metabolism of sphingolipids.

C12 Possesses the Ability to Act as a Cancer-Stop and Cancer PreventingAgent.

Ceramide (Cer), sphingosine (Sph) and Sph-1-phospate (S1P) representgeneral signaling lipids with critical roles in defense mechanismsregulating apoptotic removal of damaged cells to prevent autoimmunityand cancer development^(9, 10, 19, 22).

In eukaryotes, sphingolipids are synthesized de novo in the endoplasmicreticulum (ER) via biosynthetic pathway, in which N-acyl-spingosine orCer is a core metabolite that can be further modified to producesphingomyelins or more complex glycosphingolipids. In contrast to Cer,D-erytro-sphingosine (Sph) is formed only in a result of Cer cleavage,whereas acylation of the Sph amine group with fatty acids recycles theCer. Moreover, although both phosphorylated products of Cer and Sph, C1Pand S1P respectively, may be salvaged by dephosphorylation, S1P can beirreversibly cleaved. Once generated, these molecules become “bioactive”lipids that regulate the diverse cellular functions in a manner distinctfrom the canonical paradigm of the linear signaling pathway¹⁰. Theunique complexity of sphingolipid signaling is not only in theirmetabolic interconvertion, but also is the opposite effect of individualbioactive lipids on given pathway. For example, Sph acts as a negativeregulator of cell growth and promotes the stimuli-induced apoptoticpathways²¹⁻²⁴, while its phosphorylated counterpart SIP induces the cellproliferation and protects from apoptosis^(22, 24, 25). In fact, anumber of environmental and endogenous factors/conditions alter themetabolism of sphingolipids¹⁰. Since several of these conditions arealso associated with development and progression of metabolic diseasesand cancer, it is logical to consider that an enhance ofsphingolipid-mediated apoptotic signaling could stop the diseaseprogressing from any stage if a given agent leaves healthy cells intactwhile inducing cancerous cells to self-destruct.

In this regard, the ability of C12 to affect Sph metabolism (see aboveFIG. 2) suggested that C12 acts as a potential cancer-stop and cancerpreventing agent through selective activation of apoptosis intransformed cells or inflammation-damaged cells, such as macrophagesthat produces tumor necrosis factor (TNF), an inflammatory cytokine.Consistent with this assumption, our previously published data revealedthat C12 treatment of myeloid cells results in rapid induction ofapoptosis, although non-myeloid cells show significantly delayedkinetics and reduction of the apoptotic marker expression (see FIG. 3 inKravchenko et al., 2006; ref #12).

To further test this hypothesis, we compared sensitivity of breastcancer cell line MCF7 to apoptosis induced by TNF, C12 or theircombination. In these experiments we used a control variant of MCF7(Vector) as well as MCF7 cells stably expressing negative regulators ofapoptosis, such as Bcl-2 and CLARP; Bcl-2 is an inhibitor of theintrinsic apoptotic pathway, whereas CLARP inhibits receptor-dependentapoptotic signaling, also called the extrinsic apoptotic pathway. Thecells were incubated with TNF, C12 or their combination, and proteinextracts were analyzed by Western blot for the cleavage of PARP, abiochemical marker indicative of apoptosis²⁶. As expected, prolongincubation of control and MCF7/Bcl-2 cells with TNF induced the cleavageof PARP, whereas MCF7/CLARP cells were resistant to TNF-mediatedapoptosis (FIG. 3A). In contrast, all three cell lines showedsensitivity to C12; also, the synergy between C12 and TNF was evident inall three cell lines treated by a combination of both stimuli (FIG. 3B).

Similar experiments were conducted by using normal human bronchialepithelial cells and HeLa cell line as an example of transformed cells.We observed that both HeLa and normal cells were resistant topro-apoptotic effects of TNF (FIGS. 3C and 3D). Normal cells also showedresistance to C12 and its combination with TNF (FIG. 3D), although C12alone induced the cleavage of PARP in HeLa cells. Notably, strongsynergism was observed between TNF and C12 in HeLa cells (see FIG. 3C).Thus, these data further support our assumption that C12 inducesapoptosis in cancer cells, while normal cells are relatively resistantto C12 or its combination with TNF. In addition, two stimuli—C12 andTNF—work synergistically against cancerous cells, although normal cellsstay intact.

A Combination of TRAIL and C12 or an Analog Synergistically KillsCancerous Cells.

Among a promising candidate for cancer therapeutics is tumor necrosisfactor (TNF)-related apoptosis-inducing ligand (TRAIL). Similar to othermembers of the TNF family, TRAIL initiates apoptosis through thereceptor-mediated mechanism, also referred to as the extrinsic apoptoticpathway. In contrast to other naturally occurring pro-apoptotic ligandssuch as TNF and Fas ligand (FasL), TRAIL infusion into mice does notcause a lethal response or detectable toxicity to tissues andorgans^(27-29.) Furthermore, the potential significance of TRAIL forkilling cancer cells has been supported by studies in animal modelsdemonstrating that this cytokine possesses selective toxicity to humantumor xenografts but not normal tissues^(28, 29). However, sensitivityto TRAIL-induced apoptosis is a key factor limiting the efficacy ofTRAIL treatment, because a spectrum of sensitivity is varied indifferent malignant cells^(27, 28, 30). Furthermore, similar to normalcells, some cancer cells are also resistant to TRAIL-induced apoptosis,although the basis for the sensitivity and resistance of cells toTRAIL-mediated effects is not fully understood.

The increasing understanding of the molecular details of apoptosisindicates that tumor cells can acquire resistance to apoptosis throughinterference with either extrinsic or intrinsic apoptotic signalingpathways, which commonly accompanies with defects in cell growth controlor/and with an increase in the anti-apoptotic activity of survivalpathways such as the NF-κB and Akt signaling cascades. Indeed, mostcancer cells retain the capacity to carry out apoptosis if triggeredthrough mechanisms that can overcome such anti-apoptotic activities. Forexample, inhibition of NF-κB activity significantly increases apoptosisinduced by apoptotic stimuli³¹⁻³⁴. In addition, enhancing apoptosis alsooccurs upon activation of several intracellular non-apoptotic signalingprocesses, including the JNK pathway or endoplasmic reticular (ER)stress, known in eukaryotic cells as the unfolded protein response(UPR)¹¹. Remarkably, recent observations revealed that the UPRactivators, such as tunicamycin (Tm), thapsigargin (Tg) andRRR-α-tocopherol ether-link acetic acid analog (α-TEA), sensitize cancercells to TRAIL-induced apoptosis³⁵⁻³⁷. However, these reagents induceconstitutive and sustained activation of the UPR, which usually resultsin the induction of the mitochondria-dependent intrinsic apoptoticpathways³⁸⁻⁴⁰, even in the absence of pro-apoptotic stimuli such asTRAIL^(37, 40).

Modern understanding of the intimate associations between host and themicrobiota⁴¹, suggest that microbes have evolved subtle and selectivestrategies to collaborate with host's biology^(1, 42). Normally manybacterial products induce inflammatory processes, which are a riskfactor commonly associated with the development of cancer and autoimmunediseases^(17, 43). However, it is not a case for other bacterialmolecules. For example, our recent findings revealed that C12 possessesstrong anti-inflammatory activity¹⁷. Moreover, the realization that C12also induces the UPR prompted us to explore its potential as acomplement for anti-cancer therapy.

To test this hypothesis, we examined the effect of C12 in vitro on theactivity of TRAIL against A549 cell line derived from a cancer sample ofthe human lung. Consistent with the reported observation on A549cells²⁸(Ashkenazi A et al., 1999), in vitro addition of TRAIL (50 ng/ml)to the cultured A549 cells did not significantly affect their viabilityand growth. However, the viability of A549 cells were dramaticallyreduced when a combination of TRAIL (50 ng/ml) and C12 (1 μM) was addedto the cultured medium (FIG. 4), although the cell growth and viabilitywas practically unchanged in the presence of C12 (1 μM or 25 μM), itsunnatural stereoisomer C12R (25 μM) or a combination of TRAIL (50 ng/ml)and C12R (25 M) (FIG. 4A). Titration experiments revealed that more then50% of cells lost their viability after a 24-hours incubation in thecultured medium with 100 nM of C12 and 50 ng/ml of TRAIL (FIG. 4B).These data support our assumption that resistance to TRAIL-mediatedapoptosis might be overcome in the presence of C12.

To address whether C12 increases the pro-apoptotic effect of TRAILagainst cancerous cells, we compared responses of A549 cells to TRAIL,C12 or TRAIL+C12 by Western blot analysis for several biochemicalparameters relevant to the regulation of apoptosis and cell growth aswell as cellular stress responses. Namely, the cleavage ofpoly(ADP-ribose) polymerase (PARP) and caspase-3 were used as two commonmarkers indicative of processes induced through both the extrinsic andintrinsic apoptotic pathways^(26, 44); we also examined the cleavage ofcaspase-9 as a marker indicative of the intrinsic apoptotic pathway⁴⁵;activation of the protein kinase JNK pathway, a hallmark of themammalian stress response linked to cell growth control⁴⁶⁻⁴⁸, was testedby the analysis for the phosphorylation of JNK. To monitor the activityof C12, we analyzed the phosphorylation of eIF2α, a distinct feature ofER stress^(11, 49), and the phosphorylation of the protein kinase p38,an additional marker of the mammalian stress response⁵⁰. The results ofthese experiments revealed that the stimulation with C12 resulted inrapid and strong activation of the stress responses with similarkinetics for all three markers, although apoptotic signaling was notinduced at any time point tested (FIG. 5). In contrast, TRAIL treatmentwas certainly calm for activation of eIF2α phosporylation and othermarkers of the stress responses, although we noted barely detectable p38and JNK phosphorylation at 6-hour time point; also, we observed theexpected activation of caspase-3 leading to subsequent cleavage of PARP(see FIG. 5). Notably, substantial changes of both the apoptotic andstress response markers were evident in cells treated with a combinationof TRAIL and C12 at late time points (FIG. 5; compare the 2- and 6-hourtime points).

Similar studies on a panel of other cancerous and normal cells confirmedthese observations and demonstrated that C12 substantially sensitizeddifferent cancerous cells to TRAIL-mediated apoptosis, whereas normalcells were completely resistant (FIG. 6, FIG. 7 and Table 2), consistentwith our previous observations when TNF was used instead of TRAIL (seeabove FIG. 3). Thus, reciprocal synergism between C12- and TRAIL- orTNF-mediated signaling results in prominent pro-apoptotic effect againstcancerous cells, suggesting that the application of C12 might uncovernew anti-cancer therapeutic strategies.

Bortezomid (also known as velcade) is an anti-cancer drug that stronglysensitizes cancerous cells to TRAIL-induced apoptosis⁵¹⁻⁵³. Therefore,to assess a potential utility of C12 for anti-cancer therapy, wecompared the effects of these two compounds—(C12) and bartezomid(Bor)—on TRAIL-mediated apoptosis in cancerous A549 cells and primaryhuman hepatocytes. The comparable increase in sensitivity of A549 cellsto TRAIL was observed in the presence of either Bor or (C12) (FIG. 8A,top penal); also, similar levels of PARP cleavage (an apoptotic marker)were induced in response to a combination of TRAIL with Bor or (C12)(FIG. 7, bottom panel). Viability of normal human hepatocytes wasunchanged in the presence of TRAIL (FIG. 8B, top penal); also,TRAIL-treated hepatocytes exhibited no evidence of apoptosis (FIG. 8Bbottom panel). Similar examination of Bor-treated samples revealed thatalthough this drug alone exhibited relatively low levels of toxicitytoward hepatocytes, Bor-mediated toxicity as well as Bor-inducedapoptosis was substantially enhanced in the presence of TRAIL (see FIG.8B). In contrast, the hepatocytes were practically healthy and intact inthe presence of (C12) or its combination with TRAIL (see FIG. 8B). Thus,(C12) possesses potent anticancer activity without significant toxicitytoward non-cancerous cells.

Since (C12) is a prototypic member of the 3-oxo-AHL family, thestructure-activity-relation (SAR) investigations were warranted todetermine generic structural features of AHL, which are required for acancer-stop and cancer preventing activity as well as for activation ofthe UPR. To address this questions, a set of AHLs and their analogs weresynthesized (see List of AHLs and analogs), and the biological activityof all compounds were examined on a panel of cancerous and normal cells.The prototypic examples of biochemical data for the selected analogs areshown in FIG. 9 and FIG. 10, whereas Table 2 summarizes the SAR studiesfor all compounds. These comprehensive SAR studies identified severalanalogs (13, 15 and 16, see Table 1, below) of (C12), which possess(C12)-like effect on mammalian cells. Moreover, similar to (C12), allthree analogs strongly sensitize a number of cancerous cells toTRAIL-mediated apoptosis, suggesting that their application might leadto new anti-cancer therapeutic strategies.

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TABLE 1 Compounds Evaluated Compound LogP # Structure (est.)  1 C12

2.685  2 C12R

2.685  3

0.569  4

1.627  5

3.743  6

3.743  7

2.569  8

3.718  9

−0.514 10

3.299 11

1.856 12

−0.26 13 (12-N₃-12)

2.958 14

2.958 15 3-N₂

3.202 16 12-N₃-3-N₂

3.475 17 12-N₃-6- N₂-12

1.71 18

0.079 19

2.64 20

2.64

TABLE 2 Biological activities of compounds (naturally occurring orsynthetic derivatives of N-acylhomoserine lactones) synthesized forstructure-activity-relation studies in mammalian cells. EfficiencyEfficiency Efficiency Effect on cancer Natural (n) of PARP of the p38 ofthe UPR cells: synergy or synthetic cleavage activation activation withTRAIL or Compound (s) (%)* (%)* (%)* TNF 1 n 100 100 100 ++++ 2 s <0.1<0.1 <0.1 − 3 n <0.1 <0.1 <0.1 n.d. 4 n ~0.5 ~2.0 ~5.0 + 5n >100 >100 >100 ++++ 6 s <0.1 <0.1 <0.1 − 7 s <0.1 <0.1 <0.1 − 8 n <0.1<0.1 <0.1 − 9 n <0.1 <0.1 <0.1 − 10 s ~10.0 ~2.0 ~1.0 n.d. 11 s ~1.0~2.0 ~2.0 n.d. 12 s <0.1 <0.1 <0.1 − 13 s ~100 ~100 ~100 ++++ 14 s <0.1<0.1 <0.1 − 15 s ~30.0 ~40.0 ~40.0 +++ 16 s ~60.0 ~50.0 ~70.0 ++++ 17 s~10.0 ~5.0 ~10.0 + 18 s ~5.0 ~2.0 ~2.0 n.d. 19 s <0.1 <0.1 <0.1 − 20 n~1.0 ~2.0 ~2.0 + *The activity of a compound was normalized to those forN-(3-oxo-dodecanoyl) homoserine lactone (compound # 1, also abbreviatedin the text as (C12)).

Materials and Methods A. GENERAL METHODS

Cancer and Normal Cell Culture.

Lung cancer cell line A549 and colon cancer cell line HT29 werepurchased from ATCC. Breast cancer cell line MDA-MB-468 was receivedfrom J. C. Reed (The Burnham Institute, La Jolla, Calif.). Cell lineswere maintained in growth medium (GM): DMEM medium (4.5 g/l glucose)supplemented with 10% FBS (HyClone, Logan, Utah), L-glutamine,penicillin/streptomycin and nonessential amino acids (Invitrogen,Carlsbad, Calif.). Normal human bronchial epithelial (NHBE) cells andnormal human mammary epithelial (NHME) cells were obtained from Cambrex(East Rutherford, N.J.) and cultured as recommended by the manufacturer.Normal human colon smooth muscle cells were obtained from ScienCellResearch Lab (San Diego, Calif.) and cultured as recommended by themanufacturer. Primary cultures of human hepatocytes (freshly plated; notcryopreserved) and culture medium were obtained from Celsis In VitroTechnologies (Baltimore, Md.). Upon receipt of the cells, the medium wasgently aspirated from each well and replenished with InVitroGRO HImedium supplemented with Torpedo Antibiotic Mix (Celsis), and the plateswere kept in a 5% CO₂, at 37° C. in an incubator at saturating humidity.After 2-4 h, cells were stimulated as described in the text and figurelegends.

Mice, Bone Marrow-Derived Macrophages and Other Cell Culture.

C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor,Me.). Bone marrow-derived macrophages (BMDM) and murine embryonicfibroblasts (MEF) were prepared by using standard protocols that wereapproved by the TSRI Animal Care and Use Committee. L929/NCTC clone 929(connective tissue, mouse) cell lines were purchased from ATCC(Manassas, Va.). MEFs and L929 cell line were maintained in GM (seeabove). BMDM were cultured in 70% GM and 30% L929 conditioned medium. Ingeneral, cells (˜60-70% confluence) were incubated in fresh medium for12-14 h before stimulation.

Bacterial Culture.

Pseudomonas aeruginosa bacterial strains used in our studies were kindlyprovided by Dr. Scott A. Beatson, University of Queensland, Brisbane,Australia and include wild type Pseudomonas aeruginosa PAO1 (originallyfrom ATCC) and PAO1 ΔlasI. Bacterial strains used for control studiesinclude Acinetobacter baumannii, Escherichia coli, Staphylococcus aureusand Salmonella ryphimurium, all from ATCC.

Reagents and Standard Assay.

Recombinant human TRAIL was purchased from R&D System, Inc (Minneapolis,Minn.), and was used for cell stimulation in all experiments at aconcentration of 20 ng/ml or as indicated in the figure legends.Bacterial culture of E. coli expressing recombinant protein, a variant118-291 of mTRAIL, was used for the preparation of recombinant murineTRAIL isolated by standard ion-exchange chromatography on DE52 andhydroxyapatite. S. minnesota Re595 LPS was prepared as previouslydescribed.^(E1) All acylhomoserine lactones, includingN-(3-oxododecanoyl)-S- and -R-homoserine lactone (C12 and C12R,respectively), were synthesized and purified as previouslydescribed.^(E2) The purity was greater than 99% and was confirmed byHPLC/mass spectrometry analysis. All synthetic molecules were dissolvedin DMSO at 200× of the desired concentration and aliquots were stored at−20° C. In addition, a quantitative QCL-1,000 chromogenic Limulusamoebocyte lysate assay (BioWhittaker, Walkersville, Md.) demonstratedthat preparations of C12 were endotoxin free. Supernatant levels of TNFin the samples were measured by ELISA (BD Biosciences Pharmingen, SanDiego, Calif.). Total RNA was isolated by using TRizol reagent(Invitrogen).

Antibodies and Western Blot Analysis.

Anti-eIF2α, phospho-eIF2α(p-eIF2α), p38, p-p38 (Thr180/Tyr132), p-RelA(Ser536), p-I κBSer32/36), PARP antibodies were purchased from CellSignaling; anti-RelA, I κB α, IκBβ were from Santa Cruz Biotechnology.Cellular extracts were prepared and analyzed by Western blot assay aspreviously described.^(E3)

Data Presentation.

The data depicted in FIGS. 11-13 represent one of three or moreexperiments with each graph reflecting findings typical of multiplestudies.

General Chemistry Methods:

Reactions were carried out under a nitrogen atmosphere with dry, freshlydistilled solvents under anhydrous conditions, unless otherwise noted.Methylene chloride (CH₂Cl₂) was distilled from calcium hydride.Tetrahydrofuran (THF) was distilled from sodium-benzophenone.

Yields refer to chromatographically and spectroscopically homogenousmaterials, unless otherwise stated. Reactions were monitored bythin-layer chromatography (TLC) carried out on 0.25-mm EMD silica gelplates (60F-254) using anisaldehyde, KMnO₄ or PMA staining. Flashchromatography separations were performed on Silicycle silica gel (40-63mesh). NMR spectra were recorded on Bruker 400 MHz spectrometers andcalibrated using a solvent peak as an internal reference. The followingabbreviations are used to indicate the multiplicities: s, singlet; d,doublet; t, triplet; q, quartet; m, multiplet; br, broad.

Chemistry Materials:

(S)-α-Amino-γ-butyrolactone was purchased from Aldrich and used asreceived.

B. SYNTHETIC PROCEDURES AND CHARACTERIZATION DATA Synthesis of the3-diazirine derivative of C12 (3-N₂)

Preparation of 3.

Decanoic acid 1 (700 mg, 4.06 mmol) was added to a round-bottom flaskand dissolved in THF (5.0 mL) under nitrogen atmosphere at 25° C. CDI(725 mg, 4.47 mmol) was then added and the resulting mixture was stirredat 25° C. for 4 h. In a second round-bottom flask, t-butyl malonate (716mg, 4.47 mmol) was dissolved in THF (4.0 mL) under nitrogen atmosphereat 25° C. The flask was cooled to 0° C. and isopropyl magnesium chloride(4.5 mL, 2.0 M in THF, 8.93 mmol) was then added dropwise. The resultingmixture was stirred at 0° C. for 30 min and then warmed to 50° C. for 30min. After cooling to 0° C., the decanoic acid solution was added viacannula and stirred from 0° C.-25° C. overnight. The reaction was thenquenched with 1 M HCl (10 mL) and extracted with EtOAc (2×20 mL). Thecombined organic phases were washed with brine, dried with Na₂SO₄ andconcentrated in vacuo. The resulting residue was purified by flashcolumn chromatography (98:2 hexanes in EtOAc) to obtain 3 (950 mg, 86%yield). The characterization data for 3 matched that previouslyreported.^(E4 1)H NMR (400 MHz, CDCl₃, 25° C.): δ=3.31 (s, 2H), 2.48 (t,2H, J=7.2 Hz), 1.56-1.53 (m, 2H), 1.44 (s, 9H), 1.21-1.18 (m, 12H), 0.85(t, 3H, J=6.8 Hz).

Preparation of 4.

Compound 3 (1.34 g, 5.0 mmol) was added to a round-bottom flask at 25°C. and dissolved in ethylene glycol (910 mg, 14.75 mmol) and methylenechloride (30 mL). TMSCl (3.2 g, 3.7 mL, 29.5 mmol) was then added to thestirring solution dropwise at 25° C. The resulting mixture was stirredfor 4 days at 25° C. and then quenched with water (15 mL) and extractedwith methylene chloride (2×20 mL). The combined organic phases weredried with Na₂SO₄ and concentrated in vacuo. This protected material wasused as crude for the subsequent reaction. Crude ethyleneglycol-protected 3 was added to a round-bottom flask and dissolved inTHF (40 mL) under nitrogen atmosphere at 0° C. Lithium aluminum hydride(1.0 M in hexane, 1.5 equiv) was added dropwise, and the reactionmixture was allowed to warm to 25° C. After stirring for 4 h, thereaction was quenched with water and filtered. The filtrate wasconcentrated to ˜20 mL, 1 M HCl (20 mL) was added and the mixture wasstirred overnight at 25° C. The reaction mixture was then extracted withEtOAc (2×30 mL), and the combined organic phases were dried with Na₂SO₄and concentrated in vacuo. The resulting residue was purified by flashcolumn chromatography (3:1 hexanes in EtOAc) to obtain 4 (1.0 g, 80%yield over 3 steps). The characterization data for 4 matched thatpreviously reported.^(E5 1)H NMR (400 MHz, CDCl₃, 25° C.): δ=3.82 (t,2H, J=5.4 Hz), 2.64 (t, 2H, J=5.2 Hz), 2.39 (t, 2H, J=5.2 Hz), 1.56-1.54(m, 2H), 1.26-1.23 (m, 12H), 0.86 (t, 3H, J=6.8 Hz).

Preparation of 5.

The protocol for diazirine installation followed that previouslyreported.³ Compound 4 (489 mg, 2.44 mmol) was added to a round bottomflash equipped with a condenser and dissolved in methanol (8 mL). Liquidammonia (30 mL) was added, and the stirring solution was heated toreflux for 7 h. The resulting mixture was cooled in a dry ice-acetonebath, and a solution of NH₂OSO₃H (332 mg, 2.93 mmol) in methanol (10 mL)was added over 5 min. The cooling bath was removed, and the mixture washeated to reflux for 1 h. Following overnight evaporation of the liquidammonia, the reaction mixture was filtered, and the filter cake waswashed with methanol. The filtrate and washings were concentrated invacuo, and the crude diazirine was used as crude for the subsequentoxidation reaction. Oxidation of the crude diaziridine to diazirine 5was performed using the iodine-Et₃N method previously described,^(E6)and the compound was obtained following flash column chromatography (120mg, 23% yield). ¹H NMR (400 MHz, CDCl₃, 25° C.): δ=3.44 (t, 2H, J=6.4Hz), 1.64 (t, 2H, J=6.4 Hz), 1.37 (t, 2H, J=6.4 Hz), 1.26-1.17 (m, 14H),0.87 (t, 3H, J=6.8 Hz); ¹³C NMR (100 MHz, CDCl₃, 25° C.): δ=170.1, 52.6,49.8, 46.9, 31.9, 29.6, 29.6, 29.6, 29.3, 22.7, 19.4, 14.1; HRMS(ESI-TOF) nm/z calcd for C₁₂H₂₄N₂O [M+H]⁺ 212.1889. found 212.1318.

Preparation of 3-N₂.

The primary alcohol of diazirine 5 was oxidized to the correspondingacid using standard Jones oxidation conditions. ¹H NMR (400 MHz, CDCl₃,25° C.): δ=2.28 (d, 2H, J=4.4 Hz), 1.53-1.51 (m, 2H), 1.26-1.17 (m,14H), 0.84 (t, 3H, J=6.8 Hz); ¹³C NMR (100 MHz, DMSO, 25° C.): δ=177.3,168.9, 52.4, 46.3, 31.9, 29.6, 29.6, 29.6, 29.3, 22.7, 19.1, 14.1; HRMS(ESI-TOF) m/z calcd for C₁₂H₂₂N₂O₂ [M+H]⁺ 226.1681. found 226.1653. Theacid was converted to the corresponding acid chloride using oxalylchloride and coupled to (S)-α-Amino-γ-butyrolactone as previouslydescribed.^(E7,E8 1)H NMR (400 MHz, CDCl₃, 25° C.): δ=6.24 (app s, 1H),4.57-4.47 (m, 2H), 4.32-4.29 (m, 1H), 2.88-2.85 (m, 1H), 2.27-2.2 (m,3H), 1.53 (t, 2H, J=6.8 Hz), 1.26-1.15 (m, 14H), 0.87 (t, 3H, J=6.8 Hz);¹³C NMR (100 MHz, DMSO, 25° C.): δ=190.0, 175.4, 168.9, 66.3, 49.6,41.4, 32.7, 29.6, 29.5, 29.4, 29.2, 24.0, 14.3, 14.2 (14 found); HRMS(ESI-TOF) m/z calcd for C₁₆H₂₇N₃O₃ [M+H]⁺ 309.2052. found 309.2039.

C. BACTERIAL EXPERIMENTS Exposure of Cancer Cells to Bacterial Culture.

We utilized experimental systems in which products released by bacteriacan interact with eukaryotic targets while avoiding direct contact ofbacteria with cells in tissue culture. The inserts containing bacterialcultures were placed in close proximity to cultured lung cancer cellsthat were left untreated or treated with TRAIL (TRAIL was added directlyinto GM media at the same time). We used a custom fabricated insertprepared from cut polystyrene tubing (14 mm diameter×25 mm height) witha solvent-welded polyvinylidene fluoride membrane base (PVDF Duraporehydrophilic 0.1 m membrane, Millipore VVLP09050). To prepare theseinserts, one end of the cut polystyrene tubing is dipped into methylenechloride for 5 seconds, and the softened plastic is pressed onto thesurface of a cut section of PVDF membrane for 0.5 min. The softenedpolystyrene flows into the PVDF sufficiently to form an impervious bondwithout damaging the membrane pore structure. After allowing a few hoursfor the solvent to completely evaporate, inserts are immersed in 1%hypochlorite for 30 minutes followed by 6 rinses with sterile saline(performed in a laminar flow hood). The 14 mm diameter insert is anoptimal fit for the 24-well plate, with the internal area of the insertcovering ˜70% of the well surface.

We used the prototype 0.1 μm PVDF membrane inserts for studies in whichtissue culture cells are exposed to soluble products derived from P.aeruginosa PAO1 (wild type), PAO1 ΔlasI, S. aureus, S. typhimurium, A.baumannii and E. coli. The bacteria were grown on BHI agar plates,transferred to inoculum culture overnight followed by 1/100 dilutioninto 20 ml BHI broth in 125 ml baffled shake flasks for incubation at37° C., 250 rpm for 4-6 hr. The equal aliquots of the late log phaseculture or control BHI broth were transferred to PVDF inserts which wereplaced in 24-well plates containing target cells cultured in 0.5 ml DMEMgrowth medium.

Determination of C12 Concentration in Bacterial Cultures.

A 2-ml culture was acidified with 50 μl of HCl and 10 ml of ethylacetate was added, and the contents were mixed vigorously. The layerswere allowed to separate, and 5 ml of the ethyl acetate layer wereremoved, dried over MgSO₄, and concentrated in vacuo. The residue wasresuspended in cold methanol and centrifuged to remove the precipitate.The resulting methanol solution was analyzed by LC-MS (liquidchromatography-mass spectrometry) for C12 content. Reverse phase LC-MSanalysis (Agilent Zorbax column. 5 μm, 300SB-C8, 4.6×50 mm) wasperformed with gradients of MeCN-H2O-0.1% formic acid (from 0 to 1 min:5% MeCN, from 1 min to 9 min: gradient of 5% MeCN to 98% MeCN, and from9 to 11 min: 98% MeCN) allowing for quantification of C12 by measuringthe following ions: 298 (M+H⁺), 316 (M+H₂O+H⁺), 320 (M+Na⁺), and 338(M+H₂O+Na⁺). The concentration of C12 in the samples of P. aeruginosaPAO1 (wild type) was about 4.3 μM (P<0.001, n=5 independentexperiments).

D. MAMMALIAN CELL ACTIVATION

Cell Stimulation.

In all experiments, BMDM were stimulated with LPS (100 ng/ml), AHL(10-25 μM; usually BMDM and primary macrophages were stimulated by 25 μMof C12) or their combination as indicated in the figure legends. If notindicated, cancer and normal cells were stimulated with 40 ng/ml ofTNFα, 20 ng/ml TRAIL and 10 μM (in case of cancer cells) or 25 μM (incase of normal cells) of AHL.

E. CELL VIABILITY AND ASSESSMENT OF APOPTOSIS

Cytotoxicity was determined by using the XTT-based toxicology assay kit(Sigma, St. Louis, Miss.) in accordance with the manufacturer'sinstructions. For some experiments, to confirm the apoptotic nature oftoxicity, cells were analyzed using protocols and instructions of theAnnexin V-FITC apoptosis detection kit (Biovision, Mountain View,Calif.). In addition, caspase 3, 8 and 9 activities were determined bycolorimetric assays (R&D System), and the data are expressed as foldincreases compared to the values of untreated or control-treated cells(n=5).

F. EXPERIMENTAL REFERENCES CITED

-   (E1) Mathison. J. C.; Virca, G. D.; Wolfson, E.; Tobias. P. S.;    Glaser, K.; Ulevitch, R. J. J. Clin. Invest. 1990, 85, 1108.-   (E2) Kaufmann, G. F.; Sartorio, R.; Lee, S. H.; Rogers, C. J.;    Meijler, M. M.; Moss, J. A.; Clapham, B.; Brogan, A. P.;    Dickerson, T. J.; Janda, K. D. Proc. Natl. Acad. Sci., U.S.A 2005,    102, 309.-   (E3) Kravchenko, V. V.; Ulevitch, R. J.; Kaufmann, G. F. Methods    Mol. Biol. 2010, 692, 133.-   (E4) Langer, P.; Bellur, E. J. Org. Chem. 2003, 68, 9742.-   (E5) Kirihara, M.; Kakuda, H.; Ichinose, M.; Ochiai, Y.; Takizawa,    S.; Mokuya, A.; Okubo, K.; Hatano, A.; Shiro, M. Tetrahedron 2005,    61, 4831.-   (E6) Church, R. F. R.; Weiss, M. J. J. Org. Chem. 1970, 35, 2465.-   (E7) Kaufmann, G. F.; Sartorio, R.; Lee. S. H.; Mee, J. M.;    Altobell, L. J., 3rd; Kujawa, D. P.; Jeffries, E.; Clapham, B.;    Meijler, M. M.; Janda, K. D. J. Am. Chem. Soc. 2006, 128, 2802.-   (E8) Garner, A. L.; Yu, J.; Struss, A. K.; Lowery, C. A.; Zhu, J.;    Kim, S. K.; Park, J.; Mayorov, A. V.; Kaufmann, G. F.;    Kravchenko, V. V.; Janda, K. D. Bioorg. Med. Chem. Lett. 2011, 21,    2702.

All patents and publications, patent and non-patent, referred to hereinare incorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference in its entirety.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

What is claimed is:
 1. A pharmaceutical composition comprising an effective amount of an N-acylhomoserine lactone (AHL) analog compound of formula (I):

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15 carbon atoms having one or more diazirenyl groups, optionally having one or more carbonyl groups at positions 4 or greater on the alkyl, alkenyl or alkynyl groups, and further optionally substituted with azido, hydroxyl, or halo; or a pharmaceutically acceptable salt thereof; and, optionally, a pharmaceutically acceptable excipient.
 2. The pharmaceutical composition of claim 1, wherein the compound is of formula (II)

wherein R¹ is a linear alkyl, alkenyl or alkynyl of about 7 to 13 carbon atoms, optionally having one or more of: (j) a diazirenyl group; (k) one or more carbonyl groups; or (l) one or more independently selected azido, hydroxyl, or halo groups.
 3. The pharmaceutical composition of claim 1, further comprising a tumor modulating agent that is a TRAIL polypeptide.
 4. The pharmaceutical composition of claim 1, wherein the N-acylhomoserine lactone (AHL) analog is selected from the group consisting of:


5. A method of treating a tumor in a patient, comprising administering to the patient an effective amount of an N-acylhomoserine lactone (AHL) analog compound of formula (I)

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15 carbon atoms having one or more diazirenyl groups, optionally having one or more carbonyl groups, and further optionally substituted with azido, hydroxyl, or halo; or a pharmaceutically acceptable salt; and optionally, a pharmaceutically acceptable excipient.
 6. The method of treating a tumor in a patient of claim 5, wherein the compound is a compound of formula (II)

wherein R¹ is a linear alkyl, alkenyl or alkynyl of about 7 to 13 carbon atoms, optionally having one or more of: (g) a diazirenyl group; (h) one or more carbonyl groups; or (i) one or more independently selected azido, hydroxyl, or halo groups.
 7. The method of treating a tumor in a patient of claim 5, further comprising administering an effective amount of a tumor modulating agent that is a TRAIL polypeptide.
 8. The method of treating a tumor in a patient of claim 5, wherein the N-acylhomoserine lactone (AHL) analog is selected from the group consisting of:


9. The method of treating a tumor in a patient of claim 5, wherein the tumor is selected from the group consisting of lung, cervical, breast, and brain tumors.
 10. A compound of formula (I):

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15 carbon atoms having one or more diazirenyl groups, optionally having one or more carbonyl groups at positions 4 or greater on the alkyl, alkenyl or alkynyl groups, and further optionally substituted with azido, hydroxyl, or halo; or, a pharmaceutically acceptable salt thereof.
 11. The compound of claim 10, wherein the compound is a compound of formula (II)

wherein R¹ is a linear alkyl, alkenyl or alkynyl of about 7 to 13 carbon atoms, optionally having one or more of: (g) a diazirenyl group; (h) one or more carbonyl groups; or (i) one or more independently selected azido, hydroxyl, or halo groups.
 12. The compound of claim 10, in a pharmaceutical combination with a tumor modulating agent that is a TRAIL polypeptide.
 13. The compound of claim 10, wherein the N-acylhomoserine lactone (AHL) analog is selected from the group consisting of:


14. A method for inducing apoptosis, arrest of cell division, or inhibition of cell proliferation, in a tumor cell, comprising contacting the cell with an effective amount of an N-acylhomoserine lactone (AHL) compound, and an effective amount of a TRAIL, wherein the AHL compound is of formula (I):

wherein R is a linear alkyl, alkenyl or alkynyl of about 9 to about 15 carbon atoms having one or more diazirenyl groups, optionally having one or more carbonyl groups, and further optionally substituted with azido, hydroxyl, or halo.
 15. The method for inducing apoptosis, arrest of cell division, or inhibition of cell proliferation, in a tumor cell, of claim 14 wherein the AHL analog in the effective amount is an activator of the unfolded protein response (UPR), of endoplasmic reticulum swelling (ERS), or both, in the tumor cell.
 16. The method for inducing apoptosis, arrest of cell division, or inhibition of cell proliferation, in a tumor cell, of claim 14 wherein the compound is a compound of formula (II)

wherein R¹ is a linear alkyl, alkenyl or alkynyl of about 7 to 13 carbon atoms, optionally having one or more of: (f) a diazirenyl group; (g) one or more carbonyl groups; and (h) one or more independently selected azido, hydroxyl, or halo groups.
 17. The method for inducing apoptosis, arrest of cell division, or inhibition of cell proliferation, in a tumor cell, of claim 14 wherein the N-acylhomoserine lactone (AHL) analog is selected from the group consisting of: 